The present invention relates to microbiological diagnosis, and more specifically to a method for rapid microbiological diagnosis, and the equipment in the form of a kit, which is used to perform this diagnosis. This diagnosis method has applications in human and veterinary clinical medicine.
Microbiological diagnosis is based on physical, chemical and biological methods that have been widely developed in previous prior art.
For example U.S. Pat. No. 3,506,544 describes an electrochemical method for detecting bacteria through measurement of the decrease in polarographic content of the oxygen which circulates through an electroanalytical cell that contains two different electrodes immersed in an inoculated culture medium. This method uses great quantities of culture medium (15-18 ml) for its analysis, thus making handling of the samples difficult on a routine level.
Another method that has been used to detect microbial growth is a method using a voltaic cell source, which is based on the use of an appropriate medium with electrodes of noble metals and predetermined volumes, that generate a potential which drops at the moment of growth of the bacterium. Other equipment using this principle has been described in the scientific literature. For example, the equipment described by patent GB 83-17685 uses the same procedure. This patent describes a method of detection that uses the variation of the potential between electrodes that are in contact with fluid samples. Thus lower potentials are measured with implied higher impedance at the opening which cause a change in the measured signal due to undesirable and unavoidable noises. In most cases the system uses electrodes of noble metals or no recoverable gold plated electrodes.
An efficient and simple method for detecting microbial growth is based on the measurement of conductometric changes that occur in a suitable culture medium due to the microbial growth. According to pertinent literature, ionic movements produce a signal of conductivity measurement of the solution in a cell that indicates the conductometric value of the solution.
It is known that conductivity cells do not have total lineal behavior in their baseline scale. In addition, analysis depends on temperature. In U.S. Pat. No. 4,482,967 a detector and a method to measure the conductivity that corrects these defects is described. This reference shows equipment of high accuracy and complexity with special provisions to measure absolute values of conductivity in a gas chromatograph, with conventional cells and large volumes.
It is known that microbial growth can be detected in fluid samples using different methods, for example, by using a turbidimetric method in which the bacteria growth produces turbidity that is read by the system detector and compared with established standards. This system requires conventional optical sensors, with high quality optical receivers, sample containers of complex design to work with samples that include visual solids. (for example, antibiotic discs) A disadvantage of this method is that is does not analyze impure samples, i.e. it requires homogeneous optical samples, because of the optical complexity of the apparatus system. U.S. Pat. Nos. 3,832,532, 3,895,661 and 3,889,011 describe methods and apparati based on these principles. U.S. Pat. Nos. 4,021,120 and 3,714,445 describe devices (turbidimeters) which measure the turbidity of microorganisms in liquid mediums.
U.S. Pat. Nos. 4,021,120 and 3,714,445 describe devices based on turbidimetric principle to measure the growth of microorganisms in liquid mediums. U.S. Pat. No. 4,021,120 describes a device to monitor the growth of microorganisms in a liquid medium that contains gas. Samples are taken from the medium, using a pump that carries the sample to a degassing chamber, eliminating gas bubbles. The sample is then introduced into a calibrated chamber through which a light ray passes. The light ray strikes a photoelectric cell, producing a current that is increased by an amplifier. This indicates growth of the microorganisms. The magnitude of this current will depend on the intensity of the light ray and will be influenced by the turbidity of the medium. The sample is then pumped back to the receiving vessel to be analyzed. This method of measurement, as well as one described in U.S. Pat. No. 3,714,445, is complex from an optical and mechanical perspective; in addition, the measurement chamber and the pumps and ducts used to transfer the samples should be sterilized frequently, this makes its use difficult in routine diagnostic methods.
Patent GB 2 221 986 and U.S. Pat. Nos. 3,819,278 and 4,725,148 refer to turbidmeters that directly measure the microbial growth using the same principle of previous methods. They present optically and mechanically complex systems that need sterilization between each batch of microorganisms.
On the other hand, U.S. Pat. No. 3,832,532, also uses an conventional optically device that includes a cuvette of spectrophotometric quality, the device takes measurements using antibiotic discs included in its design as an interconnected bi-lobed chamber. After the incubation is completed, the liquid must pass to the other chamber for measurement, in order to avoid presence of the antibiotic disc during the reading step.
Thus the invention of U.S. Pat. No. 3,832,532 presents a system of operative and technical complexity and in addition has economical implications.
The present trends of microbiology make use the search of procedures that allow rapid identification of microorganisms (between 2-4 hours) in biological samples. To accomplish this, different strategies have been used, among them the use of specific enzyme markers.
According to the present state of the art, most of the biological samples cannot be used directly; isolation and growth of the microorganism must be completed before the sample can be identified, requiring 24-48 hours of laboratory time.
Infections of the urinary tract are considered one of the most prevalent among infectious illnesses.
The classic technique for detecting bacterial infections in urine requires cultivation on plates for at least 24 hours in order to discard all negative samples and to select positive ones.
Only 20% of the urine samples that arrive at the lab are positive, and from these 70% correspond to infections provoked by E.coli. Identification of E.coli saves time and resources, as only 30% of the positive samples would be isolated for identification.
According to the state of the art, identification of E.coli is accomplished mainly by two specific enzyme markers for this bacterium, xcex2-D-glucuronidase and tryptophanase enzymes, through Indol formation (Kovacks, N. Eine vereinfachte Methode zum der Nachweis der Indolbildung durch Bakerien. Z. Immunitatsforsch., 55; 311-315, 1928). 94% of all E.coli, a few Salmonellas and Shigullas show positive reaction towards xcex2-D-glucuronidase. Indol formation is positive for 99% of all E.coli; thus combination of both tests allows unmistakable identification of this microorganism.
Presently, different tests are being marketed, like BACTIDENT-E.coli and different culture mediums like FLUOROCULT-MUG, (both from MERCK DIAGNOSTICA). They are based on the above principle. In order to use them, an isolated colony from a previous isolation of the microorganism used to make the BACTIDENT identification must be taken; or the sample can be inoculated into the culture medium and grown for 24 hours; it is only possible to detect associated changes to the specific substrate transformation (FLUOROCULT-MUG).
A solid culture medium for simultaneous detection of coliform bacteria and/or E.coli in water samples and in foods is reported in the patent application No. WO 95/03424. 24 hours of incubation is required after inoculation of the plate with the sample to be evaluated. Similar procedures are followed in the Diagnostic Kit URILINE ID and the culture medium CPS ID, both from BIOMERIEUX, France. The incubation of the samples on solid medium for 24 hours is necessary before identifying the microorganism in both cases. The patent application No. WO 80/02433 refers to a procedure to identify bacteria through the combination of different tests to determine 26 bacterial enzymes; among them xcex2-D-glucuronidase and tryptophanase are useful to identify E.coli. In the present invention, bacteria should be isolated from the clinic specimens before their identification.
The objective of the present invention is to provide a system that allows detection of microbial growth early in samples obtained directly from animals, plants and their fluids, on which detection of growing microorganisms is determined through the use of micro-samples. This system is based on the detection of turbidimetric changes in a culture medium, produced by the growing microorganism. This system includes equipment, a diagnostic kit and a method designed for this purpose.
The uniqueness of the present technical solution is that it allows detection of infected samples obtained directly from the species that produce them.
Additionally, among other applications, the present invention permits obtaining the sensitivity pattern to the different antibiotics of microorganisms using previously isolated colonies or positive samples of urocultures and hemocultures, saving time required for isolation and purification processes.
In the particular case of urinary tract infections, the present invention allows discrimination of positive samples from negative ones using direct urine samples, even if contaminated with other bacteria. It can also include the simultaneous identification of those infected samples, specifically with E.coli bacterium.
Following the system of the invention, more than 1000 tested samples have shown a 95% correlation with the total count of viable cells in CLED culture medium; this method is conventionally used to detect urinary tract infection.
In the antibiogram determination, the correlation with Bauer-Kirby method is 92.4%, major errors 1.3%, and very major errors 0.4%.
In the determination of the antibiogram, the correlation with the Bauer-Kirby method has been established as a 75.8% predictive value for the sensitive antibiotics and an 85.9% for the resistant antibiotics, obtaining an overall sensitivity of 80.6%. The system guarantees a 90% effectiveness for the detection of sensitive antibodies.
The present invention provides useful information related to medical microbiological diagnosis in a short period of time. This information is very important to prevent the improper use of antibiotics, the development of microbial resistance, long hospital stays and death in the case of serious infections.
The system is characterized by its speed; a urine infection can be determined in a period of four hours and on positive samples reliable antibiogram results can be obtained.
At the same time this system is highly accurate in a significant manner. The obtained results can be verified as necessary.
From the social point of view, the system has a great importance because of the possibility of providing antibiotics in a prudent and beneficial way, as well as avoiding is long hospital stays. From the ecological point of view, the invention reduces the development of bacterial resistance. It is a highly flexible system, in which adaptation of the information can be altered according to the needs or requirements of the users. The system offers the possibility of changing the use of the antibiotics according to the particular needs.
The system of the present invention comprises equipment, a diagnostic kit and a method designed for rapid microbiological diagnosis, which can be applied to human and veterinary clinical medicine.
The equipment has been designed to work with large numbers of samples and uses not only an operative program, but also a functional interface between man and machine. It also provides an audiovisual alarm to signal readiness for a reading and to avoid operational mistakes. The software package for making measurements with this equipment can be installed in the free slot of a computer.
The equipment of the present invention includes the following devices:
A control module which is incorporated in a personal computer.
An interface card.
A peristaltic pump.
A sensor.
A calibrator.
A printer.
An ultraviolet (UV) lamp which may be optional.
The personal computer should have the following properties:
IBM Compatible, 386/486, 25-66 MHz.
RAM memory, minimum 1 megabyte.
Hard Disc, minimum 40 megabytes.
Floppy Disc 3xc2xdxe2x80x3, optional.
Display SVGA Color.
Keyboard, Mouse and Printer.
The peristaltic pump has been designed to produce circulation of the samples through the sensor. Its cassette can be set easily, allowing a regulated and uninterrupted flow and may be used independently or incorporated with the system. The pump flows at 2.0-2.6 ml/minute and is fed either 220 vac or 12 vdc. Its potential consumption is 0.5 watts.
A reader connects the sensor to a continuous microflow, which is used to detect turbidity changes due to microbial growth in previously prepared samples coming from different sources. The sample size may be of very small volumes (up to 200 microliters) and may be In movement,xe2x80x94i.e. in a moving liquid, where samples can or can not be influenced. Using the present invention it is not necessary xe2x80x9cto cleanxe2x80x9d the flow reader during measurements, thus minute samples can be measured continuously. This measurement is not influenced by turbidimetric variations of culture medium as a consequence of the changes of the samples; however the invention allows detection of small variations of medium turbidity due to microbial growth. Temperature control is not required for the measurements. It is also possible to make an unlimited number of measurements of different samples from different sources and microbial cell concentration.
Calibration of this sensor is unique as it is automatic and operates with a continuous flow of 2-2.6 ml/minute. Optic range of measurement is 0.00-2.00 McFarland units.
The present invention uses a calibrator based on nephelometric techniques and uses the McFarland scale. It joins together a direct light source and a photosensor and is adjusted through a program. The calibrator measures the turbidity in Mueller-Hinton liquid medium up to 0.2 McFarland units. Energy requirement is 5 vdc and consumption is 50 ma.
The UV lamp that is coupled optionally to the equipment allows identification of the bacterium E.coli in tested samples. The program offers a simple man to machine interface, is easy to manage and allows the selection of different options of bar-menu or keyboard function, combined with icons, as well as automatic data storage. It uses a structured interrogative language (SQL) for obtaining information and an external utilities xe2x80x98BACKUPxe2x80x99 which makes secure copies.
The program has an alarm system to be used in case of obstruction of the opening, an audiovisual alarm to control the reading time of each sample, as well as other utilities for technical maintenance.
The essential unique features of the equipment of the present invention are the turbidimetric static minireader, the microflow sensor which is fed by the peristaltic pump, and their connection to a microcomputer with a program package for acquisition, processing and creation of data bases, used to generate necessary reports.
FIG. 1 shows the general overview of the integral plan of the equipment of the present invention. As shown, this equipment comprises a turbidimetric reader of microflow (1) which is fed by a peristaltic pump (2) and adjusted to electronic equipment of high sensitivity (3) that detects turbidimetric changes of microflow (1) through a measurement method that allows use of a group of algorithms to detect small turbidimetric variations and to properly process obtained data. It is formed by a turbidimetric measurement circuit (4) connected through an interface card to a central processing unit (5). This unit receives all keyboard commands (6) and delivers the results in a display (7). It can detect smallest variations of turbidity that occur in the culture-inoculated medium in the sample that will be tested.
FIG. 2 shows a detailed design of the internal structure of the turbidimetric reader (1) where the variations of turbidity are detected. The procedure for measuring is very simple: the small opening of the reader (8) is introduced into the sample to be tested which then circulates through the reader with help of the peristaltic pump (2) of FIG. 1. This peristaltic pump works continuously throughout the measurement. First the presence of the sample is detected by obtaining a voltage value that exceeds a pre-designated value and after a specified period of time the measurement is completed. As the peristaltic pump (2) in FIG. 1 continues to operate, there is a period of time between each measurement during which air circulates through the turbidimetric reader (1). This period of time is considered as the moment in which the turbidimetric reader (1) is cleaned. It is not necessary to make the additional step of washing in order to sterilize all parts of the reader.
The measurement chamber is composed of a plastic tube (9) introduced in a glass capillary (10). The light pass (11) is the orifice diameter through which the light coming from the photoemissor (12) should travel to reach the measurement chamber (9 and 10). The intensity of the luminous radiation, transmitted through the measurement chamber (9 and 10) will depend on the turbidity grade of the sample and is measured by a photodetector (13). The radiation produced by the photoemissor (12) is stabilized by means of an electronic circuit of conventional automatic control.
FIG. 3 shows a flow diagram using the turbidimetric reader. First the presence of the turbidimetric reader is verified by means of the subroutine of detection. If present, the existence of the peristaltic pump is checked, since the pump is necessary for the operation of the turbidimetric reader and for the cleaning process subroutine; the subroutine also establishes the required operational flow. The cleaning subroutine is important as the reader""s parameters depend on the cleanliness of the measurement chamber. This aspect influences its effective life span.
After all the working parameters have been regulated, the turbidimetric reader will be ready. Any subroutine that is not functional will disable the reader. The main application of this device is directed toward the antibiogram determination of the sample, (antibiotic microorganism susceptibility), which is achieved between 2 and 6 hours, supported by a diagnostic kit designed for these purposes.
The other device that is incorporated into the equipment of the present invention is the static turbidimetric minireader mentioned before. It detects turbidity changes due to microbial growth in the sample developed in a glass vial containing liquid culture medium, one of the components of the diagnostic kit of the present invention. This vial is precisely fitted to the reading shaft of the equipment. The device is adjusted to use the vials for the direct reading of the sample; special cuvettes are not necessary to determine readings, making it possible to use in routine diagnostic mediums. The device calibrates the inoculum that is used in the diagnostic kit for detecting the antibiogram. It reports turbidity in McFarland units, according to latex patterns of NCCLS standards in an established range of measurement (0-4.0 McFarland units).
The diagnostic kit of the present invention is comprised of an 8-ml nephelometric vial that contains 4.5 ml of culture medium; the vial is made of autoclaveable borosilicate glass with a plastic cover.
FIG. 4 shows the components of the diagnostic kit of the present invention, consisting of a vial containing culture medium and the polymer, the strip support and the strip used for antibiogram determination of the tested sample. Modified sterile Mueller-Hinton Broth OXOID, pH of 7.4+/xe2x88x920.2, together with a polymer, is used as culture medium in order to follow microbial growth.
The diagnostic kit used for detecting antibiogram in a sample utilizes antibiotic discs available commercially. They can be used in conforming designs that change according to need. Antibiotic discs are organized into non-transparent strips containing two free positions for negative and positive controls, which are filled with only culture medium and inoculated culture medium respectively.
They are used to calculate the growth index in the tested samples. There are 10 to 22 additional positions where antibiotic discs could be placed.
The program created for these purposes allows the introduction of these changes in the acquisition and editing processes. The kit has high flexibility and permits adaptations according to different needs. A polymer, that could be any linear polysaccharide of structural formula CH3-CH3-CH3-N or somewhat similar with molecular mass between 50,000 and 150,000, is added to the glass vial containing culture medium for dilution, forming part of the diagnostic kit of the present invention. The incorporation of this polymer into the culture medium, at a concentration between 0.05 and 1%, allows elimination of the inhibitor effect of catabolic products accompanying the tested sample inoculum; greater growth indexes of the infection-involved bacteria are obtained in a shorter period of time, in comparison with the required time when the same culture medium is used without said polymer. This new element in the diagnostic kit reduces false sensitive results obtained in susceptibility studies, and at the same time improves the correlation with Kirby-Bauer method of reference (Bauer, A. W.; Kirby, W. M. M.; Sherris, J. C. and Turck, M. An. J. Clin. Pathol. 1966, 45, pages 493-496).
Considering that this polymer can be metabolized only by a reduced number of microorganisms which are generally not found in analysis where the present system is applied, it is inferred that the bacterial growth effect is motivated by an inhibition of the repressor agents which are present in the culture medium. For this reason the said polymer should act by absorbing the catabolites which are involuntarily incorporated together with the inoculum that is analyzed. It has been observed that during the microorganism growth measurement process, after the said polymer neutralizes these catabolites, the bactericidal activity of tested antibiotics is more specific.
One of the advantages of the present invention is the fact that the system allows diagnosis of not only previously isolated strains but also direct samples of positive hemocultures, urine, etc.
First the sample is placed in the glass vial containing the polymer and the culture medium, immediately after the turbidity is determined (t0) and this value, along with the reading time according to the adjusted number of each sample, is fixed by the employed program. The vial is then incubated between 2 and 5 hours at a temperature between 35 and 37xc2x0 C. At the end of the incubation the system emits, according to the programmed routine, a beep alarm sound and screen warning indicating that the sample should be read again. The samples that show increments higher than 0.08 McFarland units are considered positive samples.
Once positive samples are detected, the system of the present invention allows determination of their antibiogram in a very short time. For that purpose, an aliquot of the sample is transferred to a new dilution vial that contains fresh culture medium, which is distributed in the strip containing two controls (positive and negative) and from 10 to 22 antibiotic discs. After a 4-hour incubation period at a temperature of 37xc2x0 C., the strip is read by placing the microflow sensor in each microwell, following the program instructions, which selects the moment of each measurement in series. Thus the influence of the previous reading is eliminated. From the obtained density values, the growth indexes are calculated (in the controls), as well as the inhibition percentage for each sample by each antibiotic. According to the inhibition level shown by the samples, the criteria of resistant, intermediate or sensitive, are adjusted among inhibition values in a range of 60 to 100%. This means that those samples with inhibition percentage smaller than 60% can be considered resistant, those showing values between 60 and 80% can be considered sensitive at an intermediate level and those which are inhibited between 80 and 100% are considered sensitive to the antibiotic tested.
Each result is checked in order to ascertain if it is between a minimum or maximum admissible growth (satisfactory antibiogram). The obtained results and edit data of each sample automatically create the corresponding databases.
As was previously noted, when applied to urocultures, the invention permits analysis with direct samples in liquid medium, read in commercial vials, and then executes antibiogram in positive samples. This process is completed in less than 9 hours, avoiding previous steps of isolation and purification of the samples and obtaining sensitivity levels greater than 90%.
This system also allows facing generated conditions due to the contaminated samples as well as the infection caused by more than one bacterium. The contaminated condition has been overcome, adjusting the magnitude sign and the time for detecting internationally accepted infected levels ( greater than 100,000 ufc/ml). This way, contaminated samples that are not infected are excluded from further processing because their smaller bacterial levels ( less than 1000 ufc/ml) allow detection of contaminated samples only when they are infected. Taking into account the fact that the contaminated species are generally saprophytes, that, according to gram reaction, are sensitive to all antibiotics, in this particular case, it is evident that they cannot influence the detection of the resistance pattern of infected strains. These agree with the distinctive characteristics of the present invention.
In the case of infections produced by more than one bacterium, two situations could be presented. First, one of the infecting bacteria could predominate due to a greater specific growth rate after the minimum time of incubation; in this case the antibiogram will be accepted. Secondly, where both bacteria grow at the same rate, the antibiogram could show one effective antibiotic for both, or a plan of absolute resistance could be presented. In this case a new antibiotic could be tested and the sample should pass through isolation and purification procedures.
This collective analysis and rapid solution for each particular situation is possible due to the application of the reading concept in direct samples with high level of interference, in a brief amount of time and fixed aspects that characterize and distinguish the system of the present invention.
In addition to reporting detection of urinary tract infection and infecting bacterial susceptibility pattern, bacterial identification should be incorporated in order to produce a complete report. In order to achieve this goal, to the vial containing culture medium and polymer used for detection of urinary infection, two substrates that allow rapid identification of E.coli bacteria in urine might be added into the same detection vial, as has been stated before.
In the present invention, a designed culture medium that could be used in the proposed system allows detection of E.coli from infected urine samples tested after 4-6 hours of incubation time.
Proposed culture medium in the present invention include per liter, more than conventional nutritive bases included in Oxoid Mueller Hinton culture medium: Meet infusion, 300 mg ; casein hydrolyzate, 17 g. and starch, 1.5 g., substrates as MU-xcex2-glucuronide (0.1 g), L-tryptophane (1 g), and used polymer with de-repression activity (1 g). All of these components are soluble in potassium phosphate buffer 50 mM, and medium pH is adjusted between 7-7.5. The medium is then distributed by volumes of 4.5 ml into vials that are sterilized by autoclave for 20 minutes at 121xc2x0 C. The previous substrates are useful for detection of MU-1 xcex2-D-glucuronidase and tryptophanase enzymatic activity produced by E.coli. For the last enzyme, Indol detection is needed. As such, after bacterial growth is obtained in the medium, an auxiliary reactive is added for activity development (modified Kovacks chemical reactive), whose formula is paradimethylamino benzaldehyde (2 g), ethanol and concentrated chlorhydric acid (20 ml).
Signals of enzymes-substrate interaction are detected, as a first step by exposing vials with turbidity increment (positives), to a UV light source (item that optionally could be included with the equipment of the present invention), for detection of fluorescence that is generated from released xcex2-methylumbelliferone. As a second step, Indol production is tested in the same vial by addition of Kovacks modified reactive.